Kobe J. Med. Sci.,

Vol. 56, No. 5, pp. E220-E230 2010

Function of strawberry notch Family Genes in the
Zebrafish Brain Development
AI TAKANO1,3*, RIYO ZOCHI1, MASAHIKO HIBI2,TOSHIO TERASHIMA1, and YU KATSUYAMA1
1

Division of Anatomy and Neurobiology, Department of Physiology and Cell Biology,
Kobe University Graduate School of Medicine, Kobe 650-0017, Japan
2
Laboratory for Vertabrate Body Axis, RIKEN Center for Developmental Biology,
Kobe 650-0047, Japan
3
Research Fellow of the Japan Society for the Promotion of Science(JSPS)
Received 20 October 2010/ Accepted 26 October 2010

Key Words: zebrafish, brain development, strawberry notch, gene expression
ABSTRACTS
We previously reported embryonic expression pattern of strawberry notch (sbno)
family genes, suggesting involvement in brain development. However function of sbno
genes in the vertebrate development has not been known yet. Utilizing zebrafish
embryos, we experimentally examined function of sbno genes during brain
development in this report. Knockdown experiments of sbno1 and sbno2a disrupted
brain morphology, and delayed developmental alteration of gene expression. The
earliest effect of loss of function of sbno genes on the zebrafish embryogenesis that we
found here was downregulation of otx2 expression. Knockdown of sbno1 specificallyaffects regionalization along the anterior-posterior axis of the brain. These results
suggest essential roles of sbno genes in vertebrate brain development.
We carried out a microarray screening to identify novel mouse genes which are involvedin brain morphogenesis, and identified Sbno1, a vertebrate strawberry notch (sbno) familygene (3). Subsequently we cloned three sbno genes (sbno1, sbno2a and sbno2b) of zebrafish(31). Expression pattern of Sbno1 (mouse) and sbno1 and sbno2a (zebrafish) suggested
involvement of sbno genes in vertebrate brain development (3, 31), but their developmental
function has not been examined yet. Because knockdown experiments of gene function canbe easily carried out in zebrafish embryos by injection of morpholino antisense
oligonucleotide, here we examined sbno function utilizing zebrafish embryos. Injection ofmorpholinos against sbno1 or sbno2a abnormalized brain morphology, and made delay inneural gene expression. Simultaneous knockdown of sbno1 and sbno2a basically caused
severer abnormalities than that of single knockdown of either sbno1 or sbno2a, but somephenotypes were unique to one of these. These results suggest that sbno1 and sbno2a are
involved in a molecular mechanism, which assures temporal regulation of gene expression
during zebrafish brain development.

Phone: +81-78-382-5325 Fax: +81-78-382-5328 E-mail: ai0926ta@med.kobe-u.ac.jp
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FUNCTION OF SBNO FAMILY GENES IN BRAIN DEVELOPMENT
MATERIALS AND METHODSFish embryos
Wild-type zebrafish (Danio rerio) embryos were obtained from natural crosses of fishwith the AB/India genetic background. The embryos were incubated at 28.5°C in E3 embryo
medium.
Whole mount in situ hybridization
Zebrafish specimens were fixed, hybridized and stained as described previously (14).
BM purple (Roche) was used as the alkaline phosphatase substrate.
Gene knockdown by injection of morpholino antisense oligonucleotide
Fertilized eggs at 1-cell stage were injected with 1nL of 0.5mM Morpholino antisense
oligonucleotides (MOs). As controls, we injected similar amounts (8-16 ng) of Standard
Control MO (Gene Tools, LLC). MOs targeting the ATG region of sbno1 and sbno2amRNAs were designed and synthesized by Gene Tools. Two distinct MOs were prepared foreach gene and confirmed that the two MOs against the same gene cause essentially the same
results, suggesting specificity of the MOs we used in this study. The MO sequences are
MO1-1 (TCCGCAGGATCAGGATGTCTCCGCT) and MO1-2 (AACACGCTGTGCTGTC
GGTGTCCGT) against sbno1, and MO2-1 (GCATCAGGCTCCGACCAGAAACATG) andMO2-2 (GATAGTCCTCCGTGTCCATGACAAC) against sbno2a. Mainly MO1-2 andMO2-2 were used because of their stronger effectiveness than others.
Histology
Zebrafish larvae were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS).
After dehydration in acetone, specimens were embedded in a plastic resin (Technovit 8100,
Heraeus Kulzer GmbH & Co.). The plastic block was serially sectioned at 10µm thickness.
The sections were stained by hematoxylin and eosin.
RESULTS
Normal brain morphogenesis requires sbno genes
Gene knockdown (KD) experiment by morpholino antisense oligonucleotide (MO)injection was employed to examine the function of sbno1 and sbno2a in the zebrafish brain
development. To comfirm specificity of the abnormalities, we generated two species of MOagainst each gene, which are complementary to different region of cDNA sequence as
described in Materials and Methods. Because sbno1 and sbno2a exhibited the similar
expression pattern (31), we carried out not only single KD, but also double KD experimentby coinjection of MOs against sbno1 (MO1) and sbno2a (MO2). As a control experiment,
similar mount of control MO (Gene Tool) was injected, which gave no difference fromuninjected embryos in our experiments.
Injection of sbno MOs did not affect spatial and temporal pattern of cell cleavage, timingof 100% epiboly and onset of the gastrulation (Fig.3). At 50% epiboly stage, surface of theanimal side of the sbno2a-KD (Fig. 1C) and double KD (Fig. 1D) embryos became rough.
The rate of increase of number of the somite did not show any distinct difference between
control and sbno-KD embryos. Tail of double KD embryos was significantly short at the
tailbud stage (Fig. 1H). In normal embryos, the hindbrain was laterally expanded making
regional difference of the brain morphology along the anterior-posterior axis (Fig. 1I), while
this morphogenesis was not observed in sbno-KD embryos (Fig. 1J,K). The double KD
embryos exhibited severe morphological defect in the brain and the ventricle was not clearly
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A. TAKANO et alrecognized at tailbud stages (Fig. 1L). In 2day post fertilization (dpf) zebrafish embryos,
yolk of sbno-KD larvae was much bigger than that of control larvae (Fig. 1M), suggesting
disruption of organs required for absorption of nourishment. The tail was curved ventrally inthe double KD (Fig. 1P) and sbno2a-KD (Fig. 1O) larvae. The pigmentation of ventral sideof the eyes of sbno-KD larvae was poor (Fig. 1M-P). Morphology of the eyes was highly
disrupted in the double KD larvae (Fig. 1T).
Differentiation of two otoliths in the otic vesicle was observed in 2 dpf normal larvae(Fig. 1W). Although increase of number of otoliths was observed in small fraction of thesingle KD larvae, basically sbno-KD reduced the number of otoliths (Table I). Probably,
mild suppression of otolith differentiation resulted in fragmentation of the otoliths by chance.
Almost all double KD larvae did not have otolith (Fig. 1U). Thus, sbno-KD suppressed
differentiation of the otoliths.
Table I. The percentages of the appearance of number of the otoliths in each inner ear of MO injectedlarvae were summarized in the table I.

Serial sections of MO-injected 2 dpf larvae were made for detailed observation of brain
abnormalities. In single KD embryos, the ventricle was expanded and the neuroepithelium
was thinner (Fig. 2B,C), compared to the normal counterpart (Fig. 2A). Characteristic
morphogenesis was observed in the midbrain of normal larvae (Fig. 2Ac), whereas it was notobserved in sbno-KD larvae (Fig. 2Bc, Cc, Dc). Cells dissociated from the neuroepithelium
were observed in dorsal side of the ventricule of the sbno2a-KD and the double KD larvae(Fig. 2Ca, Cb). In the double KD larvae, the ventricle was narrow and the neuroepithelium
was thicker than the normal counterpart (Fig. 2D). The brain size of double KD larvae wasclearly smaller along both the dorsal-ventral and anterior-posterior axes than that of normal
larvae (Fig. 2). The surface of the eye ball of normal larvae is occupied by single layered
cells and no cell was observed inside the lens (Fig. 2Ab), but there was ectopic cells in thelens of double KD larvae (Fig. 2Db, Dc).

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FUNCTION OF SBNO FAMILY GENES IN BRAIN DEVELOPMENT
Fig. 1. Morphology of the morpholino antisense oligonucleotide-injected embryos.
Fertilized eggs were injected with non-specific control MO (A, E, I, M, Q), MO against sbno1(MO1) (B, F, J, N, R), MO against sbno2a (MO2) (C, G, K, O, S), or MO1 and MO2 (D, H, L,
P, T). The living embryos were photographed at 50% epiboly (A-D), 18-somite stage (E-L), and48 hours post fertilization (hpf) (M-T). The lateral view of the embryos are shown, where thedorsal is to the right in A-H and M-P. The dorsal view of the embryos (I-L) and larvae (Q-T) is
shown. The otoliths in the inner ear of 48 hpf larvae is shown at high magnification (U-X).

Fig. 2. Serial section of the sbno knockdown larvae
Larvae were injected with non-specific control MO (the series indicated by large letter "A"),MO against sbno1 (MO1) (the series indicated by large letter "B"), MO against sbno2a (MO2)(the series indicated by large letter "C"), or MO1 and MO2 (the series indicated by large letter"D"). The larvae embedded in plastic block were sectioned at 10μm thickness.

Effects of sbno knockdown on early genes expression
Because MO injection disrupted brain morphology, we examined expression of genes
involved in neural development. The earliest event of neural development is the neuralinduction, by which dorsal part of the ectoderm takes neural fate by function of the organizer
during gastrulating stage. Thus, we examined expression of genes that are essential for theorganizer function at the onset of gastrulation (4 hpf) by whole mount in situ hybridization.
However, obvious difference was not observed between control and sbno-KD embryos in theexpression of dharma (34) (Fig. 3 A-D) and chordin (23) (Fig. 3E-H). Expression of sox3,
one of the earliest genes expressed in the presumptive neuroectoderm (26, 27, 29, 36) was
examined at dome (4 hpf) and 80% epiboly (8 hpf) stages, and significant difference in sox3
expression was not observed between control (Fig. 3I,M) and sbno-KD embryos (Fig. 3N-T).
otx2 is also one of the conserved earliest genes expressed in the presumptive neuroectoderm(18), and essential for the normal brain development (1, 21, 24). Expression of otx2 wasdetected in the dorsal part of the animal hemisphere of the control embryos at the dome stage(Fig. 3Q). Expression of otx2 was slightly reduced in sbno1-KD embryos (Fig. 3R), very
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A. TAKANO et alfaint in sbno2a-KD embryos (Fig. 3S), and abolished in double KD embryos (Fig. 3T).
However, there was no significant difference in otx2 expression between control and KD
embryos at 80% epiboly stage (Fig. 3U-X). These observations suggest that sbno-KD delay
the onset of otx2 expression. Because dharma is a transiently expressed gene (34),
temporally normal expression of dharma in sbno-KD embryos implies that developmental
process of the knockdown embryos was not generally delayed. This is consistent to theobservations that the timing of early cell cleavage, the onset of epiboly and gastrulation wascomparable among control and sbno-KD embryos (Fig. 1). Thus, it is likely that otx2 is oneof the earliest gene affected by sbno-KD.

Fig. 3. Effects of sbno knockdown on theexpression of neural inducers andearly neuroectodermal markers.
Expression of the Spemann organizer
genes, dharma (A, B, C, D) and
chordin (E, F, G, H), and earlyneuroectodermal markers, sox3 (I-P)
and otx2 (Q-X) was detected by whole
mount in situ hybridization in thecontrol MO (A, E, I, M, Q, U), MO
against sbno1 (MO1) (B, F, J, N, R,
V), MO against sbno2a (MO2) (C, G,
K, O, S, W), or MO1 and MO2 (D, H,
L, P, T, X) injected embryos at dome
stage (4 hpf) (A-L, Q-T), and 80%
epiboly stage (M-P, U-X). The lateralview was shown except for Q-T which
are shown from animal pole of theembryos. The dorsal is to the rightexcept of Q-T, in which dorsal is to the
top.

Expression of delta and HuC genes in the brain affected by sbno knockdown
In the retinal development of Drosophila, sbno is involved in transcriptional regulation of
Delta gene (32). Thus, we examined expression of four delta genes of zebrafish. BecausedeltaA and deltaB of zebrafish are early markers for neuronal differentiation (9), we also
examined expression of HuC, the definitive neuron marker (15).
In 5-prime stage embryos, HuC expression marked the upper and lower rhombic lips, theprethalamus, the thalamus, the hypothalamus, and the telencephalon (Fig. 4A). While HuC
expression in the telencephalon was not significantly affected, that in the thalamic regions
became very faint in sbno-KD embryos (Fig. 4F, K, P). Since expression of deltaA in thedeveloping brain starts earlier than that of HuC, delta A expression is more densely observed
than that of HuC in a similar spatial pattern (Fig. 4B). Expression of deltaA was reduced in
the thalamus and abolished in the tectum by sbno-KD (Fig. 4G, L, Q). Effect of sbno-KD on
deltaB expression was similar to the case of deltaA (Fig. 4C, H, M, R). Expression deltaCand deltaD in the telencephalon of sbno-KD embryos was stronger than that in control
embryos (Fig. 4D, I, N, S). Because expression of deltaC and deltaD in the telencephalon
was gradually reduced from around 5-prime stage (n= 9; data not shown), strong expression
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FUNCTION OF SBNO FAMILY GENES IN BRAIN DEVELOPMENTof these genes in this brain region of sbno-KD embryos does not suggest enhancement ofgene expression, but it likely suggests delay of developmental change of expression of thesegenes. Expression of HuC and four delta genes indicated that the morphogenesis of the upperrhombic lip is suppressed in the sbno-KD embryos at this stage.

Fig. 4. Effects of sbno knockdown on the expression of early neuronal genes in the brain. The zebrafish
embryos injected with control MO (A-E), MO1 (F-J), MO2 (K-O), or MO1 and MO2 (P-T)
were fixed at 24 hpf and hybridized to the RNA probe against HuC (A, F, K, P), deltaA (B, G, L,
Q), deltaB (C, H, M, R), deltaC (D, I, N, S), or deltaD (E, J, O, T). Lateral view of the larvae isshown. Anterior is to the left.

Fig. 5. Effect of sbno knockdown on regionalization of the neural tube along the anterior-posterior axis.
Expression of otx2 and hoxa2 (A-C) or fgf8 and krox20 (D-F) was simultaneously detected in
the 24 hpf embyos injected with control MO (A, D), MO1 (B, E), MO2 (C, F). Lateral view ofthe larvae is shown. Anterior is to the left.

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A. TAKANO et al
Knockdown sbno1 specifically narrowed midbrain region
Since spatial expression pattern of sbno1 is different from that of sbno2a along theanterior-posterior (AP) axis (31), we investigated effects of sbno-KD on AP markers of thebrain. In 30-somite (24 hpf) stage, otx2 is expressed in the dorsal thalamus and the midbrain
regions (30), and hoxa2 is expressed in rhombomere 2, 3, 4, and 5 (11). There was not
significant difference in spatial expression pattern of these genes between control andsbno2a-KD embryos (Fig. 5A, C), while hoxa2 expressing region and posterior boundary ofotx2 were anteriorly shifted in sbno1-KD embryos (Fig. 5B). Anterior boundary of otx2 wassimilar in control and sbno-KD embryos (Fig. 5A-C). Expression of fgf8 in the isthmus andkrox20 in the rhombomere 2 and 4 was similar in control and sbno2a-KD embryos (Fig. 5D,F), whereas it was anteriorly shifted in sbno1-KD embryos (Fig. 5E). Expression of HuC(Fig. 2) also indicated anterior shift of gene expression in the hindbrain of sbno1-KD
embryos (Fig. 4F). These observations suggest that sbno1-KD narrowed otx2-expressing
region, which is compartible with the predominant expression of sbno1 in otx2 expressing
region (31).

Fig. 6. Effects of sbno knockdown on late brain gene expression. Expression of cux2 (A,B), lhx2 (C,D),
rora2 (E,F) and er81 (G,G) was examined in the larvae at 2 dpf (A,C,E) and 3 dpf (B, D, G).
The larvae were injected with control MO (indicated by small letter "a"), MO1 (indicated bysmall letter "b"), MO2 (indicated by small letter "c"), or MO1 and MO2 (indicated by small
letter "d"). Lateral view of the larvae is shown. Anterior is to the left.

Effects of sbno-knockdown on late neural genes
Our previous report (3) suggested possible involvement of Sbno1 in the laminarformation in the development of the mouse cerebral cortex, Although zebrafish forebrain
does not exhibit lamination, we expected that conserved function of Sbno family genes could
be predicted by examining expression of zebrafish genes, of which homologues are essential
for laminar development of the mouse cerebral cortex.
In the 2 dpf sbno-KD larvae, cux2 expression in the telencephalon and eyes remained
weakly, and expression in the other regions was almost abolished (Fig. 6Aa-d). Expressionof cux2 in the telencephalon and the hindbrain became weak in 3 dpf normally, whereas it
was strong in the sbno-KD larvae (Fig. 6Ba-d). Interestingly, cux2 expression in the 3dpf
sbno1-KD larvae was similar to that in the 2 dpf normal larvae (Fig. 6Aa, Bb-d). Similarities
were also observed between lhx2 and rora2 expression patterns of normal 2 dpf larvae and
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FUNCTION OF SBNO FAMILY GENES IN BRAIN DEVELOPMENT
sbno-KD 3dpf larvae. For example, expression of lhx2 and rora2in the posterior hindbrain
became very weak in the normal 3 dpf larvae (Fig. 6Ca, Da, Ea), whereas expression of lhx2
and rora2 was strongly detected in the hindbrain region of sbno-KD 3dpf larvae (Fig. 6 Dc,
Dd, Fb, Fc). In the normal 2 dpf larvae, expression of er81 was detected in habenular nucleus
and weakly in the tectum, and sbno-KD abolished er81 expression completely (data notshown). In 3 dpf sbno-KD larvae, dense expression of er81 was detected in the dorsal
telencephalon, whereas the expression in other brain regions was very faint (Fig. 6Gb-d).
DISCUSSION
In the previously study we showed that sbno1 and sbno2a are expressed in thedeveloping CNS during zebrafish embryogenesis (31). Although sbno2a and sbno2b proteins(Sbno2 homologues of zebrafish) are similar in their amino acid sequence, they exhibitedcompletely different gene expression pattern (31). In this report, single KD of either sbno2a
or sbno1 abnormalized zebrafish embryogenesis, indicating that both sbno1 and sbno2agenes are required quantitatively for normal embryogenesis. Specific effect of sbno1-KD on
the presumptive midbrain region is consistent to the fact that the presumptive midbrain
region strongly expresses sbno1. Thus, sbno1 and sbno2a have likely redundant function
biochemically, but also have unique function based on the gene expression pattern.
Possible interaction of Notch signal and sbno proteins in zebrafish development
Bray (4) suggested three different functions of Notch signal, i.e., lateral inhibition,
lineage decisions, and boundary making (induction). In Drosophila, sbno is involved in
inductive events in retinal cell differentiation (33) and in boundary formation in Drosophila
wing development (25) but not in lateral inhibition (5, 18, 20). Our results in zebrafish
experimental system are in good correspondence with these Drosophila studies as below.
The lateral inhibition event is essential for determination of proper number of neuronaldifferentiation in both vertebrates and Drosophila. Although disruption of this developmental
event is one of crucial abnormalities of Notch signal mutants, the lateral inhibition isnormaly observed in Drosophila sbno mutant (5). The zebrafish mib mutant suggests
involvement of Notch signal in lateral inhibition which is required for normal primaryneuron differentiation (12), but significant abnormality was not observed in the number ofprimary neurons marked by HuC, deltaA, or islet1 expression in sbno-KD embryos (data notshown). We previously confirmed binding between sbno1 and Su(H) proteins of zebrafish byimmunoprecipitation assay (31). Because Su(H) is required for normal lateral inhibition ofzebrafish (7), it is likely that function of Su(H) in the lateral inhibition does not involve sbno.
The defects in the somitogenesis are the characteristic phenotype observed in all of the
Notch pathway mutants of zebrafish (35). In sbno-KD embryos, formation of somite
boundaries underwent in a normal time course, but the chevron-shaped morphology of eachsomite was transiently disrupted. The otolith differentiation involves Notch signal (10, 28),and sbno-KD larvae lost the otoliths. These observed abnormalities of sbno-KD suggest
possible interaction of sbno and Notch function in some developmental events which involve
lineage decision and boundary making.
Genetic interactions between Notch signal and chromatin remodeling mechanism havebeen suggesed previously. Spt6 protein has nucleosome assembly function, affecting
function of SNF/SWI chromatin remodeling complex in yeast (8). Spt6 can bind to Notch
receptor in nematode. Interestingly, an spt6 mutant and sbno morphants of zebrafish are very
similar in terms of morphology and gene expression profile (16). Overexpression of Brg
gave similar phenotype to that of Delta-Notch mutants (2). Immunoprecipitation assay
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A. TAKANO et alindicated that SWI/SNF binds to CBF-1 (a vertebrate homologue of Su(H)) (13), and Notch
intracellular domain binds to Brm (6). Furthermore, Baf60c is required for Notch dependenttranscriptional activation of Nodal gene in mouse and zebrafish embryos (32). Thus, it isinteresting to study a possibility that sbno is involved in chromatin remodeling mechanisms.
Regulation of brain gene expression by sbno
We found that sbno-KD suppressed expression of otx2, the earliest gene to be expressedin the presumptive neural ectoderm (19, 22, 24), in the 50% epiboly stage embryos.
Because dharma, an organizer marker is very transiently expressed at this stage (34), this is agood marker to show that sbno-KD is specifically affected on normal gene expression.
Normal expression of the organizer markers suggests that the neural induction by the dorsal
mesoderm takes place normally in sbno-KD embryos and a possibility that sbno is directly
involved in transcriptional regulation of otx2.
Loss of function of sbno genes not only delay onset of gene expression, but also delays
reduction of expression of genes. The delay of brain gene expression in the sbno-KD was 2 or
3 hours at gastrula stage, whereas it became more than 24 hours at 3 dpf. Taking all of theseobservation into account, sbno may be required for progression of developmental change ofgene expression.
ACKNOWLEDGEMENTS
We thank members of the Division of Anatomy and Developmental Neurobiology, Kobe
University Graduate School of Medicine and the Laboratory for Vertebrate Body Axis, CDB
RIKEN for their support throughout this study. This work was supported by a research grantfrom Japan Society for the Promotion of Science to A.T, and grant-in-aid from the Ministryof Education, Culture, Sports, Science, and Technology of Japan to Y.K.

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